Supporting Online Material
Materials and Methods
Seedling growth and measurements
Seeds were surface sterilized and plated on growth medium (GM)
without sucrose as described by Huq and Quail (2002) (S3). The
red and far-red light sources have been described by Wagner et
al. (1991) (S4). Fluence rates of various lights were measured
using a spectroradiometer (model LI-1800, LiCor, NE). The T-DNA
tagged seeds were obtained from the Arabidopsis Biological
Resource Center (Columbus, OH) and Torrey Mesa Research
Institute, (San Diego, CA) (S5-S6). Hypocotyl length
measurements are performed as described by Huq and Quail (2002)
(S3).
Transformation of Arabidopsis and transgenic plant analysis
The PIF1 open reading frame from the full-length cDNA was
amplified using PFU Turbo polymerase (Stratagene) with forward
and reverse primers, which included restriction sites for
cloning. The resulting fragments were cloned into the pKF111
vector (S7) in both orientations and sequenced. These constructs
were introduced into GV3101 (MP90) Agrobacterium and used for
transformation of wild type Col-O by the floral dip method (S8).
Transgenic seeds were plated on GM-Suc plates containing 5 µg/ml
of glufosinate-ammonium (Riededel-de Haen, Germany), and the
2
resistant seedlings were transplanted to soil and grown in the
greenhouse.
In vitro co-immunoprecipitation assay
For in vitro co-immunoprecipitation experiments, all proteins
were expressed from T7 promoters in the TnT in vitro
transcription/translation system (Promega) in the presence of
35S-methionine. The DNA constructs for expressing phyA, phyB,
GADPIF4, and GADPIF3, are described by Huq and Quail (2002)
(S3). The GAD:PIF1 vector was constructed by PCR amplification
of GAD and PIF1 separately, followed by three fragment ligation
into a pET17b vector (Invitrogen), and confirmed by sequencing.
In vitro co-immunoprecipitation experiments, sample preparation
and quantification were performed according to Huq and Quail
(2002) (S3).
RNA isolation and Northern blotting
Total RNA was isolated from 4-day-old seedlings of wild type
and pif1 mutants using Qiagen RNeasy Plant Mini Kit (Qiagen,
Valencia, CA). Full-length PIF1 ORF was used as a probe to
detect message levels in the wild type, pif1-1 and pif1-2 mutant
backgrounds.
Subcellular Localization of PIF1
For subcellular localization, full-length PIF1 open reading
frame was amplified using PCR primers containing restriction
3
sites (Cla I - Sal I), and fused to the C-terminus of GUS in the
TEX3 vector digested with Cla I - Sal I (S3). The resulting GUS-
PIF1 fusion construct was transiently transfected into onion
epidermal cells and stained for GUS activity according to Huq
and Quail (2002) (S3).
Construction of plasmids and transactivation assays
For Gal4 DNA binding domain (GBD) fusions, the PIF1 open
reading frame was cloned as a SmaI – KpnI fragment into pMN6
(S9) in frame to GBD in front of a full-length cauliflower
mosaic virus (CaMV) 35S promoter. pMN6 alone was used as a
negative control. For detection, the Dual – Luciferase system
was used (Promega). The LUC+ gene in pGLL was driven by a
chimeric promoter containing two copies of the lac operator and
four copies of the Gal4 binding site fused upstream of a minimal
35S promoter (-98) fragment. Renilla lucerifase driven by a full
length CaMV 35S promoter was used as internal control (pRNL).
Three-day-old etiolated Arabidopsis Col wild type or phyB-9 or
phyA-211 mutants were grown on 0.8% GM-S plates on filter paper.
Before bombardment the hypocotyls were laid flat to create a
continuous square of seedlings. 10 µg of effector plasmids, 10 µg
of pGLL reporter and 0.5 µg of pRNL internal control were co-
precipitated in 10 mM spermidine and 1.25 M CaCl2 onto 1 µm gold
particles, washed, dried in ethanol, split in two and delivered
4
to two tissue plates by particle bombardment at 1100 psi using a
Dupont Biolistic particle delivery system. The tissue was then
treated with 15 min far-red light (730 nm, 20 µmolm-2s-1) and then
transferred either to darkness, continuous red (660 nm, 13 µmolm-
2s-1) or continuous far-red (730 nm, 20 µmolm-2s-1) light for 16
hrs. Alternatively, the seedlings were exposed to a pulse regime
of either 9x 5 min red pulses (55 µmolm-2s-1) or 9x 5 min red
pulse followed by a 5 min far-red pulse (40 µmolm-2s-1) every 2
hours.
The tissue was extracted in LUC extraction buffer (200 mM
NaPO4 pH 7.8, 4 mM EDTA, 2 mM DTT, 5% glycerol, 1 mg/ml BSA, 2 mM
PMSF and 1xcomplete protease inhibitor cocktail (Roche) at 3x
w/v. Debris was separated by centrifugation at 14,000 rpm and 20
µl of the supernatant were used to measure firefly and Renilla
luciferase activity. In each experiment eight replica plates
were used for each effector and treatment.
Supporting Figures
Supplementary Material Figure 1: PIF1 is not necessary for
phytochrome-mediated hypocotyl suppression. A) Mean hypocotyl
lengths of wild type Col, transgenic overexpression lines of
PIF1, two pif1 T-DNA insertion lines and their wild type
siblings grown under dark (D), red (Rc, 32 µmolm-2s-1) and far-red
light (FRc, 17 µmolm-2s-1) conditions for four days. B) Visible
5
phenotypes of seedlings grown under the same conditions as
described in (A).
Supplementary Material Figure 2: Photobleaching of pif1 mutants
under prolonged incubation in light. A) In vivo low temperature
(77K) fluorescence emission spectra of cotyledons of 4 day old
etiolated seedlings of wild type (WT) and pif1 that were kept in
the dark or exposed for 10 min, 6 h, 15 h and 24 h to white
light (125 µE m-2 sec-1). Single cotyledons were cut from each
seedling after the light treatment and frozen immediately in
liquid nitrogen for the measurement of the emission spectra. The
four major peaks represented by downward arrows shows the
positions of free protochlorophyllide (Peak 1),
protochlorophyllide bound with protochlorophyllide
oxidoreductase enzyme (POR) (Peak 2), chlorophyll/photosystem II
(Peak 3) and photosystem I (Peak 4), respectively. The
excitation wavelength was 433 nm and the emission was recorded
between 600 and 800 nm. B) In vitro fluorescence emission
spectra of acetone extracts of WT and pif1 mutant seedlings
grown in the dark for 4 days and exposed to white light for 15
and 24 h, respectively. Extracts of 10 seedlings were used for
each time point. The excitation wavelength was 433 nm and the
emission was recorded between 600 and 700 nm. The low
temperature fluorescence spectra were recorded as described
6
earlier (S1). Extraction and measurement of tetrapyrroles are
according to op den Camp et al. (2003) (S2).
Supplementary Material Figure 3: Cotyledon areas of wild type,
pif1, phyA and phyB mutant seedlings. A) Wild type and pif1
seedlings have similar cotyledon area. Cotyledon area of two-
day-old dark-grown, or dark-grown seedlings transferred to white
light (50 µmolm-2s-1) for 5 hrs were measured for wild type and
pif1 mutants. Digital pictures were taken from at least 30
cotyledons of each genotype and area was measured using NIH
ImageJ software. B) Wild type, phyA and phyB mutant seedlings
have similar cotyledon areas. Digital pictures were taken for
three-day-old dark-grown, or dark-grown seedlings transferred to
white light for 5 hrs and cotyledon area was measured as
described in A.
Supplementary Material Figure 4: PIF1 is localized to the
nucleus. Top: Schematic representation of GUS only and GUS-PIF1
fusion constructs used in transient assays in onion epidermal
cells. Bottom: Left pair of panels show GUS staining for GUS-
PIF1 and GUS only control. Middle pair of panels shows DAPI
staining for nuclei in the left panels. Right pair of panels
shows superimposition of the GUS and DAPI staining. GUS-PIF1, β-
glucuronidase fused to the amino-terminus of the full-length
PIF1 protein.
7
Supplementary Material Figure 5: PIF1 binds to the G-box DNA
motif present in various light-regulated promoters. A) PIF1
binds to the G-box in a sequence-specific manner. One µl of TnT-
expressed PIF1 was used in each lane indicated (+). Thirty
thousand cpm of labeled probe was used in each lane. The binding
conditions, amount of probe used, and the gel compositions are
described in Huq and Quail, 2002 (S3). FP, free probe. B) G-box-
bound PIF1 does not bind to phyA and phyB. Numbers on the top
are µl of each component added in each lane. The binding
conditions, amount of probe used, and the gel compositions are
described in Huq and Quail, 2002 (S3).
0
2
4
6
8
10
12
14 D Rc FRc
Col
PIF1Ox 1
P IF 1Ox2
pif1- 1
pif1- 2
WTSib ling
WTS ibling
Col
PIF 1Ox1
PIF1Ox2
pif1- 1
p if1-2
WTSibling
WTS ib ling
MMeeaannHHyyppoocc oottyylllleenngg tthh ss
(( mmmm))
AA BB
RRcc
FFRRcc
DD
Figure S1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2600
615
629
644
658
673
687
702
716
731
745
760
774
789
R.F.U
pif DD
wt DD
pif DDL 10'
wt DDL 10'
pif DDL 6h
wt DDL 6h
pif DDL 15h
wt DDL 15h
pif DDL 24h
wt DDL 24h
in vivo
1
2
3
4
0
5
10
15
20
25
600
608
615
623
630
638
645
653
660
668
675
683
690
698
pif light 15h
wt light 15 h
pif light 24h
wt light 24h
in vitro
Figure S2
A
B
Wavelength (nm)
Wavelength (nm)
Rela
tive
Fluo
resc
ence
Uni
tsRe
lativ
e Fl
uore
scen
ce U
nits
Figure S3
PIF1GUS
GUS
GUS DAPI SUPERIMPOSED
GUS-PIF1
GUS
Figure S4
FP
PIF1
PIF1TnT
Probe G-wt G-mut cold
competitorA
+ ++++++
1 2 3 4 5 6 7 8 9
RFR
FP
- 1 1 1 1 1 - - - -- - - - - - 1 1 1 -- - - - 4 4 - 1 1 -- - 4 4 - - - - - -
TnT
phyBPIF3PIF1
Light
PIF3+PfrBPIF3PIF1
- 4 - - - - - - - 4phyA
RFR RFR
1 2 3 4 5 6 7 8 9 10
B
Figure S5
13
Supporting References:
S1. N.N., Lebedev, P., Siffel, A.A. Krasnovskii, Photosynthetica
19, 183 (1985).
S2. R.G.L. op den Camp et al. Plant Cell 15, 2320 (2003).
S3. E. Huq, P. H. Quail, EMBO J. 21, 2441 (2002).
S4. D. Wagner, J. M. Tepperman, P. H. Quail, Plant Cell 3, 1275
(1991).
S5. A. Sessions et al., Plant Cell 14, 2985 (2002).
S6. J.M. Alonso et al., Science 301, 653 (2003).
S7. M. Ni, J. M. Tepperman, P. H. Quail, Cell 95, 657 (1998).
S8. S. J. Clough, A. F. Bent, Plant J. 16, 734 (1998).
S9. M. Ni, K. Dehesh, J. M. Tepperman, P. H. Quail, Plant Cell
8, 1041 (1996).